B6.Cg-Ptprc^a Pepc^b Gt(ROSA)26Sor^{tm1.1(CAG-cas9*,-EGFP)Fezh} Tg(TcraTcrb)425Cbn/ShrJ

Stock number: 040737
Product name: OT-2 CD45.1 Cas9
Strain synonyms: OT-2 CD45.1 Rosa26-Cas9 , OT-II CD45.1 Rosa26-Cas9
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

OT-2 CD45.1 Cas9 mutant mice express CD45.1 and carry the OT-2 transgene designed to recognize ovalbumin peptide residues 323-339 and a Rosa26-Cas9 knock-in allele that constitutively expresses cas9 endonuclease and EGFP. When used in combination with single guide RNAs, the Rosa26-Cas9 allele allows for editing of single or multiple mouse genes. This mutant mouse line is useful for tracking gene knock-out CD4⁺ T cell responses in vivo or ex vivo.

Detailed description

This mouse line is a C57BL/6J congenic strain that carries the differential Ptprc^a pan leukocyte marker, commonly known as CD45.1 or Ly5.1, that is used widely in transplant studies (see Stock No. 002014). It also carries the OT-2 transgene that contains mouse alpha-chain and beta-chain T-cell receptor subunits gene (see Stock No. 004194). This transgenic T cell receptor was designed to recognize ovalbumin peptide residues 323-339 (OVA323-339) in the context of (CD4 co-receptor interaction with MHC class II). This results in CD4⁺ T cells that primarily recognize ovalbumin peptide residues 323-339 when presented by the MHC class II molecule. Mice also contain a Rosa26-Cas9 knock-in allele that constitutively express CRISPR associated protein 9 (Cas9) endonuclease and EGFP in a widespread fashion under the direction of a CAG promoter (similar to Stock No. 026179). When used in combination with single guide RNAs, the Rosa26-Cas9 allele allows for editing of single or multiple mouse genes. This mutant mouse line is useful for tracking gene knock-out CD4⁺ T cell responses in vivo or ex vivo.

Due to the presence of the OT-2 transgene, there is a significant reduction in the number of CD8⁺ T cells.

The donating investigator has made other similar mouse lines available including OT-1 CD45.1 Rosa26-Cas9 mice (Stock No. 040288) and P14 CD45.1 Cas9 mice (Stock No. 040736).

Development

To create this mutant mouse line, Dr. Arlene Sharpe (Harvard Medical School) crossed Rosa26-LSL-Cas9 knock-in mice on a C57BL/6J congenic background (see Stock No. 026175) to Zp3-Cre mice (Stock No. 006888) to remove the floxed-STOP cassette in the female germline. The Zp3-Cre allele was then bred out of the mice. The resulting Rosa26-Cas9 mice were then crossed to B6 CD45.1 mice (Stock No. 002014) and OT-2 transgenic mice (Stock No. 004194). All mouse lines used were previously bred to C57BL/6J (Stock No. 000664) for at least 5 generations. OT-2 CD45.1 Cas9 mice were maintained on a C57BL/6J genetic background by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize B6 CD45.1 oocytes.

Allele

Symbol: Tg(TcraTcrb)425Cbn
Name: transgene insertion 425, Frank Carbone
MGI accession ID: MGI:3046083
Generation method: Transgenic
Attributes: Inserted expressed sequence
Synonyms: OT2; OT-2; OTII; OT-II; OT-II alphabeta TCR; OTII TCR; OT-II.2; TCR-OT-II.2a; TCRTg; Tg(TcraTcrb)425-2Cbn; TgN(OT-II.2a); TgN(TcrOT-II)
Marker symbol: Tg(TcraTcrb)425Cbn
Marker name: transgene insertion 425, Frank Carbone
Marker synonyms: OT2; OT-2; OTII; OT-II; OT-II alphabeta TCR; OTII TCR; OT-II.2; TCR-OT-II.2a; TCRTg; TgN(OT-II.2a); TgN(TcrOT-II)
Chromosome: UN
Strain of origin: C57BL/6 x B6.C-H2^bm1
Expressed genes: Tcra,T cell receptor alpha chain,mouse, laboratory; Tcrb,T cell receptor beta chain,mouse, laboratory
Molecular note: The transgene was generated by co-injection of two constructs created by separate cloning into a T-cell receptor expression vector the PCR-amplified cDNAs encoding mouse alpha-chain and beta-chain T-cell receptor subunits expressed by a CD4⁺ T-cell hybridoma designated Clone 1.1. The reconstituted receptor pairs with the CD4 coreceptor and is specific for chicken ovalbumin residues 323-339 in the context of H2-Ab1 (I-A^b).
General note: Transgenic mice express the mouse alpha-chain and beta-chain T-cell receptor that pairs with the CD4 coreceptor and is specific for chicken ovalbumin 323-339 in the context of I-A b. Homozygous transgenic mice are viable and fertile. In these mice there is a four-fold increase in the CD4 to CD8 peripheral T-cell ratio, and lymph node T-cells demonstrate a dose-dependent proliferative response to the specific ovalbumin ligand. Phenotypes for the two sublines (OT-II.2)425-2 and 426-6 are described as identical. (J:87876)

Allele

Symbol: Pepc^b
Name: b variant
MGI accession ID: MGI:4819861
Generation method: Not Applicable
Synonyms: Pep3^b
Marker symbol: Pepc
Marker name: peptidase C
Marker synonyms: Dip-1; Pep3; Pep-3
Chromosome: 1
Strain of origin: Not Applicable
Molecular note: The Pepc gene controls electrophoretic variation of peptidase C (PEP-C; EC 3.4.11.-) of red blood cells. The allele Pepc^b determines a faster migrating band and is found in the CBA/J and A/He strains.

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-cas9*,-EGFP)Fezh}
Name: targeted mutation 1.1, Feng Zhang
MGI accession ID: MGI:5583838
Generation method: Targeted
Attributes: Reporter; Endonuclease
Synonyms: constitutive Cas9; Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}; Rosa26-Cas9
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: cas9,CRISPR associated protein 9,; GFP,Green Fluorescent Protein,
Site of expression: EGFP and cas9 will be expressed in a widespread fashion under the direction of a CAG promoter
Molecular note: The targeting vector inserted into the locus was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm. Cre-mediated recombination removed the floxed STOP cassette.

Allele

Symbol: Ptprc^a
Name: a variant
MGI accession ID: MGI:4819849
Generation method: Not Applicable
Synonyms: CD45.1; Ly5^a; Ptprc^SJL
Marker symbol: Ptprc
Marker name: protein tyrosine phosphatase, receptor type, C
Marker synonyms: B220; CD45; CD45R; GP180; IMD105; LCA; L-CA; LY5; Ly-5; Lyt-4; RT7; T200
Chromosome: 1
Strain of origin: Not Applicable
Site of expression: Widely expressed on all adaptive and innate immune cells.
Molecular note: Ptprc^a is found in strains SJL/J, STS/A, and DA. Ptprc^b is found in strains C57BL/6, C3H/An, DBA/2, AKR, and many others (J:13367, J:12054, J:12077, J:8603). Twelve nucleotide differences between the a and b alleles have been identified. These base substitutions correspond to five amino-acid changes within the extracellular domain of the encoded protein. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat sequences (J:22485).

Note that the allele designations originally described were reversed in 1987 (J:8603); all publications prior to 1987 show SJL/J, STS/A, and DA as having the b allele and the C57BL/6J group as having the a allele (J:22341).